bioRxiv preprint doi: https://doi.org/10.1101/2021.03.02.433159; this version posted March 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Short Note

Uncovered genetic diversity in mabouia (Reptilia: ) from Island reveals uncertain sources of introduction

Catarina Rato1*, Beatriz Martins1,2, Ricardo Rocha1,3†, Iolanda Silva-Rocha1†

1 CIBIO, Research Centre in Biodiversity and Genetic Resources, InBIO, Universidade do Porto, Campus de Vairão, Rua Padre Armando Quintas nº7, Vairão 4485 - 661, Vila do Conde, . 2 Departamento de Biologia, Faculdade de Ciências da Universi dade do Porto, R. Campo Alegre, s/n, 4169 - 007, Porto, Portugal. 3 CIBIO- InBIO, Research Center in Biodiversity and Genetic Resources, Institute of Agronomy, University of Lisbon, Lisbon, Portugal † These authors have contributed equally to this work

* Corresponding author: [email protected]

Total number of words in the whole manuscript: 3,274

Total number of words in the abstract: 151

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36 Abstract 37 Hemidactylus mabouia is one of the most widely distributed within its genus. It was 38 first reported to Madeira in 2002 and the first individuals were considered to have originated 39 from Cape Verde. Almost 20 years later, we found that H. mabouia has substantially expanded 40 its distribution and can now be found >10 km away from , where it was first reported. 41 Based on a 12S phylogenetic analysis and using 29 individuals from Funchal and Câmara de 42 Lobos we found that Madeira actually harbours two distinct lineages of H. mabouia: one 43 exclusively South American and another widespread in America and Africa. However, the lack 44 of genetic diversity typical of this species outside its native range and the obtained phylogenetic 45 pattern prevent us to infer possible introduction routes or sources. Our study emphasizes that 46 authorities should remain vigilant regarding the arrival of other non-natives and act to prevent 47 their establishment as soon as they are detected. 48 49 Keywords: Hemidactylus mabouia, Madeira Island, biological invasions, conservation, 50 phylogenetic analysis 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68

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69 The rate of species’ translocation to areas outside their native geographical range is increasing 70 with human globalization, considerably impacting native biotas (Simberloff et al, 2013). 71 are particularly prone to biological invasions, either by being one of the most often 72 introduced groups, or by being notably sensitive to the impacts of invaders (Kraus, 73 2009). The introduction of species to island ecosystems is particularly problematic, due to the 74 limited capacity of insular native species to compete with newcomers, elude predators and 75 handle diseases, inherent from their evolutionary insular history (Novosolov et al, 2013; 76 Whittaker and Fernández-Palacios, 2007). Hemidactylus Gray, 1845 is one of the most species 77 rich genera within the Gekkonidae family, currently comprising about 164 recognized species 78 (Uetz et al, 2020). These have their native distribution throughout most of tropical Asia 79 and Africa, the more arid regions of northeast Africa and southwest Asia, the Mediterranean 80 region and , which they reached by natural transmarine colonization (Kluge, 81 1969). They have recently colonized, both naturally and anthropogenically, other areas of the 82 Americas and the West Indies, Australia and several islands of the Indian, Pacific and Atlantic 83 Oceans (see references in Carranza and Arnold, 2006). Their success as human-assisted 84 colonizers is partly associated with the synanthropic habits of several Hemidactylus species, 85 allied with their relatively small size and cryptic nature (Hoskin, 2011). The tropical house 86 Hemidactylus mabouia is one of the most successful invaders within the genus, and it can now 87 be found in both the Old and New Worlds, as well as in many Atlantic islands, such as Cape 88 Verde (Jesus et al, 2001; although classified later as in Vasconcelos 89 et al, 2013 following Rocha et al, 2010), the Gulf of Guinea Islands (Jesus et al, 2005) and 90 Madeira (Jesus et al, 2002). The successive introductions and absence of genetic differentiation 91 within H. mabouia over its extensive range, precludes the assessment of the ultimate 92 geographic origin of this species (Carranza and Arnold, 2006). Nonetheless, the great genetic 93 diversity observed on Mayotte island in the Comoros, and the relationship to northeast 94 Madagascar populations assigned to H. mercatorius, suggest that the widespread haplotypes 95 originated in East Africa, and from there spread westwards, across tropical Africa reaching 96 several Atlantic islands and large parts of tropical America (Carranza and Arnold, 2006). 97 Additionally, based on the deep divergence observed between African and Malagasy 98 specimens, Vences et al (2004) began to refer to all H. mabouia populations in Madagascar as 99 Hemidactylus mercatorius (Gray, 1842). Moreover, results from Rocha et al (2005) clearly 100 show that some specimens of H. mabouia from the Gulf of Guinea cluster with one H. 101 mercatorius clade from northern Madagascar and the Comoros. Because Boumans et al (2007) 102 argued in favour of the native status of H. mercatorius across Madagascar, Rocha et al (2010)

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103 proposed that insular populations of H. mabouia should be recognized as H. mercatorius, and 104 the status of East African populations should remain unchanged as H. mabouia, pending further 105 studies. Clearly, the complex phylogeographical patterns and of the putative 106 “species-complex” H. mabouia/mercatorius is far from being understood. 107 In this study, we focus on the genetic affinities of H. mabouia specimens from Madeira Island, 108 in an attempt to infer the geographical origin of the introduced populations. The volcanic 109 archipelago of Madeira is located ca. 520 km from the African coast and is comprised by the 110 islands of Madeira, and Desertas (fig. 1). Almost 20 years ago, Jesus et al (2002) 111 reported the first three specimens in Madeira, captured in the island’s capital Funchal. These 112 were assigned as H. mabouia based on their genetic similarity with other sequences 113 available at the time on GenBank (12S and cytb). Through the analysis by eye of the alignment, 114 the authors concluded that the Madeiran specimens were most similar to previously sampled 115 individuals from the Cape Verdean island of São Vicente. Here, apart from collecting 116 specimens from an additional locality in Madeira, the total number of analysed individuals is 117 substantially larger, allowing the detection of possible within island intraspecific genetic 118 variation. Additionally, through a phylogenetic analysis, we use available H. mabouia 119 sequences from the species’ known distribution to infer possible routes and sources of 120 introduction of Madeira’s populations of tropical house geckos. 121 122 A total of 29 individuals assigned in the field to H. mabouia were collected from two distinct localities in Madeira, 123 namely Funchal (N=16) and Câmara de Lobos (N=13). In Câmara de Lobos, some individuals were collected in 124 the city centre, while others captured in a nearby industrial area. 125 Tissue from tail tip muscle was collected from each individual and preserved in 96% ethanol. Genomic DNA was 126 extracted using an Easy-Spin Kit (Citomed). A fragment of the 12S rRNA gene was amplified by Polymerase 127 Chain Reaction (PCR) using the primers L1091 and H1478 published by Kocher et al (1989). Amplification of 128 the 12S mtDNA fragment was carried out in a 25 µl volume, containing 0.16 µM of each primer, 1U of Taq DNA 129 polymerase (MyTaq HS Mix, Bioline), and approximately 100 ng of template DNA. PCR consisted of an initial 130 denaturation at 95°C for 10 min, followed by 40 cycles that included a denaturation step at 95°C for 30 sec, 131 annealing at 54°C for 30 sec and extension at 72°C for 30 sec. A final extension was conducted for 10 min. All 132 successful amplifications were sent to CTM (CIBIO-InBIO) for purification and Sanger sequencing. This same 133 protocol was also employed on two H. mercatorius specimens from Madagascar, with codes ACP1143 134 (Sahamalaza) and ACP2350 (Isalo). 135 A total of 35 12S sequences of H. mabouia were retrieved from GenBank and added to the dataset. Geographic 136 location of each population from Madeira is depicted in fig. 1, and detailed information about the locality and 137 GenBank accession codes is presented in Table 1. GenBank accession codes for the used H. mercatorius are 138 MW665155 and MW665156. The obtained sequences were imported into the software Geneious Prime®

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139 (v.2020.2.4 Biomatters Ltd.) where the alignment was performed using MAFFT v.7.017 (Katoh and Standley, 140 2013) under the default parameters. Phylogenetic analysis based on the 12S mitochondrial fragment was 141 performed under a Bayesian Inference (BI) method, using Gekko gecko as the outgroup, and several other 142 representatives of the genus Hemidactylus, such as H. mercatorius, H. longicephalus, H. greefi, H. brasilianus, 143 H. palaichthus and H. platycephalus, known to be closely related to H. mabouia (Carranza and Arnold, 2006; 144 Gamble et al, 2011). In order to determine the best fitting nucleotide model, we used the software PartitionFinder 145 v.2 (Lanfear et al, 2017), considering branchlengths = linked and modelselection = BIC. The software BEAST 146 v.1.8.4 (Drummond and Rambaut, 2007) was used for the BI topology. Analyses were run twice for 25x106 147 generations with a sampling frequency of 100. Models and prior specifications applied were as follows (otherwise 148 by default): Strict Clock and Coalescent with Constant Population Size. Convergence for all model parameters 149 was assessed by examining trace plots and histograms in Tracer v.1.7 (Rambaut et al, 2018) after obtaining an 150 effective sample size (ESS) > 200. The initial 10% of samples were discarded as burn-in. Runs were combined 151 using LogCombiner, and maximum credibility trees with divergence time means and 95% highest probability 152 densities (HPDs) were produced using Tree Annotator. Trees were visualized using FigTree v.1.4.0 (Rambaut, 153 2009). 154 155 According to the obtained 12S phylogenetic topology, the genetic diversity of H. mabouia is 156 represented by four clades, with H. mercatorius as its sister taxon (fig. 1). Nevertheless, most 157 of these relationships are not highly supported. While this pattern within H. mabouia is not a 158 novelty (see Carranza and Arnold, 2006), the genetic diversity found in Madeira is. 159 Surprisingly, apart from the specimens already identified by Jesus et al (2002) as related to 160 individuals of H. mabouia from Cape Verde, this study reveals the existence of three 161 individuals belonging to a new lineage in Madeira, related exclusively to South American 162 specimens. Only individuals captured in Câmara de Lobos, both in the city and industrial area, 163 where assigned to this new lineage. Though Câmara de Lobos is mostly a traditional fishing 164 town with no international harbour, its industrial park is one of the island’s most active 165 commercial hubs, thus a highly probable entrance point of this new lineage of H. mabouia into 166 the island. Yet, the exact geographic origin of these animals is unclear as this lineage is 167 genetically uniform and widespread across Central and South America. Likewise, since Brazil 168 harbours both clades detected in Madeira (fig. 1), it precludes to disentangle if the Madeiran 169 population of H. mabouia results from a single (i.e. same origin) or multiple introductions (i.e. 170 distinct origins). At least in part of the Brazilian range of H. mabouia both lineages occur in 171 sympatry (Rio Grande do Norte State; Carranza and Arnold, 2006). As such, the most 172 parsimonious hypothesis would be that the specimens from Madeira have a Brazilian source, 173 which would imply a single colonization event. However, this is a mere hypothesis that 174 although plausible, cannot be confirmed by our results.

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175 During our fieldwork we observed that H. mabouia is now well established in Madeira and that 176 over the last 20 years the species has expanded from Funchal, where it was first detected (Jesus 177 et al, 2002), to Câmara de Lobos. In both localities we observed numerous immature 178 individuals and pregnant females, suggesting a healthy population. Yet, our observations 179 highlight the need for an island-wide survey to assess the species’ distribution. Little is known 180 also about the current range of another non-native gecko species occurring in Madeira, the 181 Moorish gecko Tarentola mauritanica, which was first reported 8 km East of Funchal in 1993 182 (Báez and Biscoito, 1993). Yet, our 2020 fieldwork, shows that T. mauritanica has greatly 183 expanded its distribution through the south cost of the island, and it can be found >20 km away 184 from the locality where it was first recorded. 185 The negative consequences of introduced species, including non-native geckos, on native 186 biodiversity are increasingly recognized (Doherty et al, 2016; Perella and Behm, 2020). 187 Hemidactylus mabouia has been observed to compete with numerous species of diurnal and 188 nocturnal geckos, including H. angulatus in Cameroon (Böhme, 1975), Gymnodactylus 189 darwinii in Brazil (Teixeira, 2002; Zamprogno and Teixeira, 1997), Phyllodactylus martini and 190 Gonatodes antillensis in Curaçao and Bonaire (Van Buurt, 2006), and Gonatodes vittatus and 191 Thecadactylus rapicauda in Venezuela (Fuenmayor et al, 2005). Furthermore, H. mabouia also 192 preys on a wide diversity of arthropods (Iturriaga and Marrero, 2013), potentially impacting 193 native arthropod taxa in the species’ non-native range. So far, no study has addressed any 194 potential effects of H. mabouia on the native fauna of Madeira. Yet, the island is home to a 195 diverse arthropod fauna with numerous narrow range endemic species (Boieiro et al, 2014), 196 which can be at risk as they might become part of the diet of the introduced geckos. 197 Over the last three decades the terrestrial fauna of Madeira has increased from one, the 198 endemic Madeira wall-lizard Teira dugesii, to at least four species due to the arrival and 199 establishment of the geckos T. mauritanica (Báez and Biscoito, 1993) and H. mabouia (Jesus 200 et al 2002) and of the brahminy blind snake Indotyphlops braminus (Jesus et al, 2013). The 201 Mediterranean chameleon Chamaeleo chamaeleon, the Peters's Rock Agama Agama 202 picticauda (Wagner et al, 2012) and the Cape Verdean skink Chioninia fogoensis (Clemens 203 and Allain, 2020) have also been reported to the island, however, the existence of established 204 populations pends confirmation. Still, the rate of arrival and establishment is alarming and as 205 such, local authorities should be vigilant regarding new arrivals and act to prevent their 206 establishment as soon as they are detected. Likewise, efforts should be made to avoid that the 207 already established species spread to the other islands of the archipelago and to the nearby 208 Selvagens Archipelago, home to the endemic Selvagens gecko Tarentola boettgeri bischoffi.

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209 Overall, our study uncovered unexpectedly high genetic diversity for H. mabouia in Madeira, 210 but the pattern of phylogenetic relationships prevents us to ascertain if the island’s population 211 results from a single or multiple introductions. Still, from a conservation point of view, H. 212 mabouia and other introduced species in Madeira deserve careful monitoring, and phylogenetic 213 studies such as the one presented here can offer important insights regarding the geographic 214 origins of introduced taxa, a key aspect for the implementation of action plans to stop the flow 215 of non-native species. 216 217 Acknowledgments 218 CR is supported by a postdoctoral contract from FCT, Portugal (DL57/2016/CP1440/CT0005) 219 and RR by a postdoctoral fellowship from ARDITI – Madeira’s Regional Agency for the 220 Development of Research, Technology and Innovation (M1420-09-5369-FSE-000002). Field 221 and lab work were supported by the project PTDC/BIA-EVL/27958/2017 (to CR) from FCT. 222 The authors would like to acknowledge Angelica Crottini for providing samples of H. 223 mercatorius. Specimens’ collection and tissue sample transport were performed under the 224 permits 05/IFCN/2020 and 03/IFCN/2020 provided by the Forests and Nature Conservation 225 Institute from the Madeiran Autonomous Region. 226 227 References 228 Báez, M., Biscoito, M. (1993): First record of Tarentola mauritanica (Linneus, 1758) from the 229 island of Madeira (NE Atlantic). In: First symposium of fauna and flora of the Atlantic 230 islands, p., Funchal, Madeira. 231 Böhme, W. (1975): Zur Herpetofaunistik Kameruns, mit Beschreibung eines neuen Scinciden. 232 Bonner Zoologische Beiträge 26: 2-48. 233 Boieiro, M., Aguiar, A., Aguiar, C., Borges, P., Cardoso, P.M., Crespo, L., Menezes, D., 234 Pereira, F., Rego, C., Silva, I., Silva, P. (2014): Madeira the Biodiversity pearl: valuing 235 the native habitats and endemic life forms, Sociedade Portuguesa de Entomologia. 236 Boumans, L., Vieites, D.R., Glaw, F., Vences, M. (2007): Geographical patterns of deep 237 mitochondrial differentiation in widespread Malagasy reptiles. Molecular 238 Phylogenetics and Evolution 45: 822-839. 239 Carranza, S., Arnold, E.N. (2006): Systematics, biogeography, and evolution of Hemidactylus 240 geckos (Reptilia: Gekkonidae) elucidated using mitochondrial DNA sequences. 241 Molecular Phylogenetics and Evolution 38: 531-545.

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275 Kluge, A.G. (1969): The evolution and geographic origin of the New World Hemidactylus 276 mabouia-brookii complex (Gekkonidae, Sauria). Miscellaneous Publications Museum 277 of Zoology University of Michigan 138: 1-78. 278 Kocher, T.D., Thomas, W.K., Meyer, A., Edwards, S.V., Pääbo, S., Villablanca, F.X., Wilson, 279 A.C. (1989): Dynamics of mitochondrial DNA evolution in animals: amplification and 280 sequencing with conserved primers. Proceedings of the National Academy of Sciences 281 USA 86: 6196-6200. 282 Lanfear, R., Frandsen, P.B., Wright, A.M., Senfeld, T., Calcott, B. (2017): PartitionFinder 2: 283 new methods for selecting partitioned models of evolution for molecular and 284 morphological phylogenetic analyses. Molecular biology and evolution 34: 772-773. 285 Novosolov, M., Raia, P., Meiri, S. (2013): The island syndrome in lizards. Global Ecology and 286 Biogeography 22: 184-191. 287 Perella, C.D., Behm, J.E. (2020): Understanding the spread and impact of exotic geckos in the 288 greater region. Biodiversity and Conservation: 1-26. 289 Rambaut, A. (2009): Figtree [Computer Software]. http://tree.bio.ed.ac.uk/software/figtree/. 290 Rambaut, A., Drummond, A.J., Xie, D., Baele, G., Suchard, M.A. (2018): Posterior 291 summarisation in Bayesian phylogenetics using Tracer 1.7. Systematic Biology 292 syy032: doi:10.1093/sysbio/syy032-doi:10.1093/sysbio/syy032. 293 Rocha, S., Carretero, M.A., Harris, D.J. (2005): Diversity and phylogenetic relationships of 294 Hemidactylus geckos from the Comoro islands. Molecular Phylogenetics and Evolution 295 35: 292-299. 296 Rocha, S., Carretero, M.A., Harris, D.J. (2010): On the diversity, colonization patterns and 297 status of Hemidactylus spp.(Reptilia: Gekkonidae) from the Western Indian Ocean 298 islands. The Herpetological Journal 20: 83-89. 299 Simberloff, D., Martin, J.L., Genovesi, P., Maris, V., Wardle, D.A., Aronson, J., Courchamp, 300 F., Galil, B., García-Berthou, E., Pascal, M., Pyšek, P. (2013): Impacts of biological 301 invasions: what’s what and the way forward. Trends in Ecology and Evolution 28: 58– 302 66 303 Teixeira, R.L. (2002): Aspectos ecológicos de Gymnodactylus darwinii (Sauria: Gekkonidae) 304 em Pontal do Ipiranga, Linhares, Espírito Santo, sudeste do Brasil. Boletim do Museu 305 de Biologia Mello Leitão 14: 21-31. 306 Uetz, P., Freed, P., Hošek, J. (2020): The . http://www.reptile-database.org; 307 accessed on November 18, 2020.

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308 Van Buurt, G. (2006): Conservation of amphibians and reptiles in Aruba, Curaçao and Bonaire. 309 Applied Herpetology 3: 307-321. 310 Vasconcelos, R., Brito, J.C., Carranza, S., Harris, D.J. (2013): Review of the distribution and 311 conservation status of the terrestrial reptiles of the Cape Verde Islands. Oryx 47: 77-87. 312 Vences, M., Wanke, S., Vieites, D.R., Branch, W.R., Glaw, F., Meyer, A. (2004): Natural 313 colonization or introduction? Phylogeographical relationships and morphological 314 differentiation of house geckos (Hemidactylus) from Madagascar. Biological Journal 315 of the Linnean Society 83: 115-130. 316 Wagner, P., Bauer, A.M., Wilms, T.M., Barts, M., Böhme, W. (2012): Miscellanea 317 accrodontia: notes on nomenclature, taxonomy and distribution. Russian Journal of 318 Herpetology 19: 177-189. 319 Whittaker, R.J., Fernández-Palacios, J.M. (2007): Island Biogeography: Ecology, evolution, 320 and conservation. 2nd Edition., Oxford University Press. 321 Zamprogno, C., Teixeira, R.L. (1997): Hábitos alimentares da lagartixa-de-parede 322 Hemidactylus mabouia (Reptilia, Gekkonidae) da planície litorânea do norte do 323 Espírito Santo, Brasil. Revista Brasileira de Biologia 58: 143-150. 324 325 326 327

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328 Tables 329 330 Table 1. Information regarding the specimens of Hemidactylus mabouia from Madeira used in 331 this study. Specimen Code Locality Geographic coordinates GenBank Accession Code DB31344 Funchal 32.644349, -16.915567 MW665176 DB31345 Funchal 32.644349, -16.915567 MW665178 DB31346 Funchal 32.644349, -16.915567 MW665180 DB31405 Funchal 32.644349, -16.915567 MW665161 DB31428 Funchal 32.644349, -16.915567 MW665182 DB31432 Funchal 32.644349, -16.915567 MW665166 DB31524 Funchal 32.644349, -16.915567 MW665169 DB31525 Funchal 32.644349, -16.915567 MW665173 DB31526 Funchal 32.644349, -16.915567 MW665177 DB31527 Funchal 32.644349, -16.915567 MW665179 DB31533 Funchal 32.644349, -16.915567 MW665181 DB31536 Funchal 32.644349, -16.915567 MW665162 DB31537 Funchal 32.644349, -16.915567 MW665165 DB31548 Funchal 32.644349, -16.915567 MW665184 DB31549 Funchal 32.644349, -16.915567 MW665170 DB31550 Funchal 32.644349, -16.915567 MW665174 DB31553 Câmara de Lobos (village) 32.647043, -16.973176 MW665157 DB31559 Câmara de Lobos (village) 32.647043, -16.973176 MW665163 DB31564 Câmara de Lobos (village) 32.647043, -16.973176 MW665158 DB31552 Câmara de Lobos (industrial) 32.647220, -16.969386 MW665167 DB31554 Câmara de Lobos (industrial) 32.647220, -16.969386 MW665171 DB31555 Câmara de Lobos (industrial) 32.647220, -16.969386 MW665175 DB31556 Câmara de Lobos (industrial) 32.647220, -16.969386 MW665159 DB31557 Câmara de Lobos (industrial) 32.647220, -16.969386 MW665185 DB31558 Câmara de Lobos (industrial) 32.647220, -16.969386 MW665160 DB31560 Câmara de Lobos (industrial) 32.647220, -16.969386 MW665164 DB31561 Câmara de Lobos (industrial) 32.647220, -16.969386 MW665183 DB31562 Câmara de Lobos (industrial) 32.647220, -16.969386 MW665168 DB31563 Câmara de Lobos (industrial) 32.647220, -16.969386 MW665172 332 333 334 335

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336 337 Figure Legends 338 339 Figure 1. a) Bayesian phylogenetic tree derived from the 12S rRNA fragment and using Gekko 340 gecko as outgroup. The stars next to the nodes represent values of posterior probabilities higher 341 than 95%. The symbol  stands for genetic diversity; b) map depicting the geographic position 342 of the Madeira Archipelago on a global scale, and in more detail the location of the two sampled 343 populations (Funchal and Câmara de Lobos) in Madeira Island. 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368

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13 Figure Click here to access/download;Figure;Figure1.jpg bioRxiv preprint (which wasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.It doi: https://doi.org/10.1101/2021.03.02.433159 made availableundera CC-BY-NC-ND 4.0Internationallicense ; this versionpostedMarch2,2021. . The copyrightholderforthispreprint